Innate immune signaling pathways are initiated when host pattern recognition receptors (PRRs) recognize incoming microbes, resulting in the induction of cytokines and antiviral type-I interferons (IFN-I). These signaling pathways are regulated by different molecular processes, including the ubiquitin (Ub) system. The goal of this proposal is to use influenza virus (IAV), which causes serious seasonal epidemics, as a model system to study the role of poly-Ub chains in activation of novel PRRs. This knowledge will help design approaches to improve antiviral responses. Ubiquitination of proteins is a post-translational modification process, which is critical in many cellular functions, including immunity. Ub has seven lysines, each of which can be conjugated by another Ub to form a poly-Ub chain. In general, proteins covalently attached to poly-Ub chains linked through lysine 48 (K48) of Ub are targeted for degradation by the proteasome, whereas protein modification with K63-linked poly-Ub chains has non-proteolytic activating functions. Unanchored poly-Ub chains that are not covalently attached to any protein have also been shown to activate kinases in innate immune signaling. To identify other cellular factors that may be regulated by non-covalent interactions with unanchored poly-Ub chains in vivo, we developed a novel approach to isolate unanchored poly-Ub chains from lungs of IAV-infected mice, and identified three RNA helicases that interact with these unanchored poly-Ub chains in vivo. Two helicases, DHX8 and DHX16, are known factors involved in pre-mRNA splicing, however whether they are involved in innate immunity or if their activity is regulated by unanchored poly-Ub is not known. Therefore, there is a gap in knowledge regarding the function of unanchored poly-Ub chains in regulation of mRNA splicing and innate immunity. In this proposal we will focus on DHX16. Our preliminary data suggest that DHX16 recognizes IAV RNA and that unanchored poly-Ub stimulates its ATPase activity. Therefore, our hypothesis is that DHX16 acts as a PRR for IAV RNA to induce antiviral innate immunity, and that unanchored K48 poly-Ub regulates its splicing activity and/or innate immune signaling. Since DHX16 localizes in the nucleus where IAV replicates, this would be the first example of a nuclear PRR and regulation of splicing by unanchored poly-Ub. In Aim 1, we will demonstrate that DHX16 acts as a PRR for IAV RNA and that unanchored poly-Ub regulates its function. We will identify the precise IAV RNA sequence that interacts with DHX16 and demonstrate that they interact directly, using in vitro and in vivo approaches. In Aim 2, we will determine the mechanism by which unanchored poly-Ub chains and IAV RNA regulate DHX16-mediated innate immune function and whether it depends on splicing or innate signaling. The outcome of our studies will elucidate a novel molecular mechanism of host defense against viruses using the Ub system and uncover a new mechanism of virus recognition by the host. This knowledge may be used to design approaches to improve antiviral responses.